1. Field of the Invention
Methods for the isolation of human progesterone or progestin receptor (hPR) from cells and the use of this hPR for antibody production are described. This antibody production includes both polyclonal and monoclonal antibody specific for the human progestin receptor and methods for the detection and measurement of human progestin receptor using the antibodies produced.
2. Background of the Invention
The present invention relates generally to the isolation of the human progesterone or progestin receptor and the use of this receptor protein in the production of antibody molecules. More specifically, this invention provides novel antibody preparations which have specific reactivity with the hPR and provide improved test methods and reagents for the detection and quantification of hPR proteins by means of immunological reactions.
It has been determined that certain tissues, notably certain human breast cancer tissues, are steroid hormone "dependent" in the sense that systemic deprivation of supportive hormones will result in regression of tissue growth and cell proliferation. As one example of this dependence, bilateral adrenalectomny can effect striking remission of advanced breast cancer in postmenopausal women and similar remissions are observed after hypophysectomy. Estrogen deprivation by surgical ablation of tissue responsible for estrogen production and/or endocrine additive therapy afford the most effective treatments presently available for advanced breast cancer. Unfortunately, less than one-half of the premenopausal patients and even a smaller fraction of postmenopausal patients respond to this type of therapy--indicating that breast cancer tissue is not always of the cellular type which is estrogenic hormone dependent. Consequently, it is significant to the prognosis and treatment of human breast cancer to be able to ascertain whether excised tumor tissue of a breast cancer patient is comprised predominantly of estrogen dependent cell types. On the basis of such information, a reasonable ablation response prediction may be made. Surgical removal of estrogen producing glands for the purpose of estrogen deprivation may be restricted to those patients most likely to be helped by the procedure. Correlatively, other breast cancer patients can be spared the trauma of essentially useless surgery and may be placed immediately into alternative therapeutic programs such as radiation or chemotherapy.
Progesterone receptors are specific progesterone binding proteins that are specific for binding progesterone or other progestins and are involved in the stimulation of progesterone specific biological responses in certain tissues. These responsive target tissues are activated by the binding of the progesterone to the progesterone receptor resulting in the progesterone receptor complex becoming tightly bound to the chromatin of the cell, thereby resulting in the ability of the cell to synthesize certain types of RNA. This regulation of gene expression in eukaryotic cells by progestins and other steroid hormones involves interaction of the specific intercellular receptor proteins with both steroid hormone and the genome, resulting in the activation of specific sets of responsive genes (1).
A result of the interaction between a hormone receptor and a steroid hormone is a change in DNA synthesis, RNA synthesis and production of proteins involved in the regulation of cell proliferation, differentiation and physiological function in diverse tissues. The hormone estrogen induces the progesterone receptor protein, whose synthesis appears to be transcriptionally regulated by the binding of estrogen receptor to chromatin. In addition, such steroid hormones and their receptors appear to be involved in the regulation of abnormal growth in various tumors and tumor cell lines (2). Data from several laboratories (3) suggest that steroid hormone action may involve the binding of the hormone directly to an intracellular receptor molecule, which is weakly associated with nuclear components in the absence of the ligand (steroid). Binding of the hormone ligand to its receptor results in the conversion of the steroid-receptor complex to a form which now associates with high affinity to one or more nuclear components. What is known about steroid receptors has come from the use of radiolabeled hormones (4) and hormone analogs to detect, quantify and characterize those intercellular steroid binding receptor proteins principally found in reproductive tissues. Although distinct steroid and DNA-binding domains have been postulated to exist for all steroid receptor proteins, including the progesterone receptor, little data is available on the detailed structure, composition and chemical properties of the subunits which bind both steroid hormones and DNA, and virtually nothing is known about the involvement of other non-steroid-binding components involved in the tissue response to steroid hormones.
Heretofore, the presence of steroid hormone dependent tissue in mammary tumor samples has principally been determined by quantitative detection of steroid hormone receptors in the sample through radiochemical assay. According to one such procedure, radioactive (e.g., tritiated) progesterone is added to the cytosol--or supernatant fraction--of a homogenized tissue sample, and the tritiated progesterone reversibly combines with any hPR protein present in the cytosol. The specimen is then subjected to low-salt, sucrose density gradient ultracentrifugation, and the receptor-progesterone complex, being a large molecule, sediments with a characteristic velocity. A radioactive count can be used to quantify the complex. This procedure is carried out in the presence and in the absence of an inhibitor of the desired specific binding in order to identify and exclude any binding that is non-specific. The above analytical technique requires use of rather sophisticated, costly and uncommon ultracentrifugation apparatus, the operation of which requires a high degree of skill on the part of the laboratory worker. Other methods employed for receptor assays have similar limitations. [See, e.g., Korenman et al., J. Clin. Endocrinol. & Metab. 30, 699-645 (1970)]. As a result, despite the exceptional usefulness of quantitative detection of the hPR in prediction of response to endocrine therapy, the utilization of prior radiochemical assays is limited by scientific, geographic, and economic considerations.
The production of monoclonal antibodies to steroid hormone receptors has recently been reviewed by E. Milgrom in Pharmac Ther., Volume 28, 389 (1985). The production of sensitive immunoassays for human steroid receptor proteins has been described by Nolan et al. in Current Controversies In Breast Cancer, University of Texas Press, 1984. The application of immunochemical techniques to the analysis of estrogen receptor structure and function has been described by G. L. Greene in Biochemical Actions Of Hormones, Volume XI, Chapter 8 (1984). The importance of receptors in the management of breast cancer has been shown by Hawkins in the British Journal Of Hospital Medicine, p. 160, September 1985.
The clinical utility of determining whether a cancer is hormone responsive, has been shown by correlations of patient responses to endocrine therapies with the human estrogen receptor (hER) and the human progesterone receptor (hPR) content of breast cancers. These studies have clearly established the importance and utility of the biochemical hER and hPR assays. While only 25-30% of unselected breast cancer patients obtained objective remission of metastatic disease following various endocrine therapies, more than 75% of the patients whose cancers contained both hER and hPR benefit (5), whereas there is only a 33% response rate if only one of the two steroid receptors is present (6). Moreover, those patients whose primary cancers are hER/hPR-rich are more likely to have a longer disease-free interval than those cancers that lack either receptor (7, 8). Data from several clinical studies (9-11) indicate that the hPR (as an end-product of estrogen action) is an even better marker than the estrogen receptor for favorable prognosis and endocrine responsiveness in breast cancer. Previously the receptor-binding assays in which hER and hPR were measured was by saturational binding of radio-labeled steroid hormones, or their analogs to the appropriate receptor protein. This assay method requires considerable laboratory skill and effort and the availability of immuno-diagnostic procedures would greatly facilitate the determination of the presence of steroid receptors.
It has been recognized that immunochemical techniques for hPR detection would, if available, provide a simpler and less costly analytical procedure and be susceptible to more widespread clinical use. However, the art has heretofore not been provided with any definitive demonstration that antibodies to hPR could be generated and effectively employed in the detection of hPR in human tissue and cells. Indeed, the difficulty in isolating hPR has created doubt that it would ever be successfully developed.
Other steroid receptor antibody-dependent assays have been developed. The generation of well-defined hER antibodies was first reported by Greene et al. (12). Polyclonal antibodies have been raised against partially purified preparations of glucocortoid receptors (13-15), calf and human estrogen receptor (16, 17), and progesterone receptors isolated from rabbit and guinea pig uterus (18, 19), and chick oviduct (20). In addition, autoimmune antibodies to androgen receptor have been found in human serum (21). In some cases antibodies will bind to corresponding receptors from other species. However, all antibodies directed against steroid binding subunits appear to be hormone specific. Monoclonal antibodies have been prepared against estrogen receptors (22-24), the glucocortoid receptor (25-27, 28), and rabbit progesterone receptor (28). The rabbit progesterone receptor antibody was reported to cross-react the hPR but only with a much lower affinity. Monoclonal antibodies have been prepared against pututative non-hormone binding forms of the B-subunit (30) and A-subunit (31) of chicken oviduct PR, although the relationship between these B and A "antigens" and the steroid binding subunits have not been established. Both polyclonal (32) and monoclonal (33) antibodies have been prepared against proteins that appear to be closely associated with steroid receptors, but which themselves will not bind hormones.